Human T-lymphotropic retroviruses (HTLV-I, HTLV-II, AND HIV) encoded protein factors that positively activate viral gene expression and replication. The transactivator protein of HTLV-I, p40x (TAT-I), is a nuclear protein of 40 Kdal in size that positively stimulates transcription from the HTLV-I long terminal repeat sequences. Three 21 base-pair repeats in the HTLV-I LTR function as a p40x dependent enhancer element that activates transcription in an orientation and position independent manner. This proposal aims at understanding the biochemical mechanism through which p40x activates transcription. Special emphasis will be placed on the following approaches: I. Molecular Genetic Approaches: This system involves using protoplast fusion to deliver the p40x protein expressed in E. coli to mammalian cells that harbor integrated copies of the HTLV-I LTR-CAT DNA. The chromatin configuration surrounding the HTLV-I LTR in the presence or absence of p40x will be studied. Mutations in the 21 base-pair repeat sequences will be created to assess the effect of (1) single base changes, (2) altered spacing of the repeats, (3) various subdomains in the 21 base-pair repeat, on basal level transcription and trans-activation by p40x. II. Biochemical Approaches: p40x protein and cellular factors that interact with p40x and/or the 21 base-pair repeats will be purified and characterized biochemically. Attempts will be made to establish in vitro systems to study the role of p40x in trans-activation. Biochemical properties of the relevant factors from HeLa cells and HeLa cell lines that constitutively express p40x will also be examined. The long range goal of this proposal is to understand the mechanism of p40x directed trans-activation at the molecular level.